Stabilizers for hemostatic products

ABSTRACT

A hemostatic product that includes a dextran support, at least one hemostatic agent and at least one hemostatic agent stabilizer. The at least one hemostatic agent is selected from the group consisting of thrombin and fibrinogen. The at least one hemostatic agent is associated with the dextran support. The at least one hemostatic agent stabilizer is associated with the at least one hemostatic agent.

REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Nos.61/548,258, which was filed on Oct. 18, 2011; 61/548,260, which wasfiled on Oct. 18, 2011; 61/548,261, which was filed on Oct. 18, 2011;61/585,303, which was filed on Jan. 11, 2012; 61/589,060, which wasfiled on Jan. 20, 2012, the contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates generally to products having hemostaticcharacteristics. More particularly, the invention relates to stabilizersfor use in hemostatic products.

BACKGROUND OF THE INVENTION

The body's natural response to stem bleeding from a wound is to initiateblood clotting via a complex process known as the coagulation cascade.The cascade involves two pathways that ultimately lead to the productionof the enzyme thrombin, which catalyzes the conversion of fibrinogen tofibrin.

Fibrin is then cross-linked to form a clot, resulting in hemostasis. Forwounds that are not severe, and in individuals that have nocountervening conditions, the body is usually able to carry out thisprocess efficiently in a manner that prevents excessive loss of bloodfrom the wound. However, in the case of severe wounds, or in individualsin whom the clotting mechanism is compromised, this may not be the case.

For such individuals, it is however possible to administer components ofthe coagulation cascade, especially thrombin and fibrinogen, directly tothe wound to bring about hemostasis. Bandaging of bleeding wounds isalso a usual practice, in part to isolate and protect the wounded area,and also to provide a means to exert pressure on the wound, which canalso assist in controlling bleeding.

While these methods may be carried out satisfactorily in cases of mildtrauma or under conditions of “controlled” wounding (e.g. surgery), manysituations in which such treatments are most needed are also those inwhich it is the most difficult to provide them. Examples of such woundsinclude, for example, those inflicted during combat or unanticipatedwounds that occur as the result of accidents. In such circumstances,survival of the wounded individual may depend on stopping blood lossfrom the wound and achieving hemostasis during the first few minutesafter injury. Unfortunately, given the circumstances of such injuries,appropriate medical intervention may not be immediately available.

In particular, the treatment of penetrating wounds such as bullet woundsor some wounds from shrapnel is problematic. This is due to thedifficulty in placing a hemostatic product and/or therapeutic agents atthe actual site of injury, which includes an area that is well below thebody surface and difficult or impossible to access using conventionaltechniques.

Jiang et al. in Biomacromolecules, v. 5, p. 326-333 (2004) teacheselectrospun dextran fibers. Agents associated with the fibers (e.g. BSA,lysozyme) are directly electrospun into the fibers. The fibers may alsoinclude other polymers electrospun with the dextran.

Jiang et al. in Journal of Biomedical Materials Research Part B: AppliedBiomaterials, p. 50-57 (2006) discloses electrospun fibers that are acomposite of poly(c-caprolactone) as a shell and dextran as a core.These fibers provide the slow release of agents (bovine serum albumin,BSA) that are also electrospun into the fibers.

Smith et al., U.S. Pat. No. 6,753,454, discloses electrospun fiberscomprising a substantially homogeneous mixture of a hydrophilic polymerand a polymer that is at least weakly hydrophobic, which may be used toform a bandage. The bandage may comprise active agents (e.g. dextran).However, the disclosed fibers are not readily soluble in liquid.

MacPhee et al., U.S. Pat. No. 6,762,336, teaches a hemostatic multilayerbandage that comprises a thrombin layer between two fibrinogen layers.The bandage may contain other resorbable materials such as glycolic acidor lactic acid based polymers or copolymers. Neither electrospun fibersnor dextran fibers are taught as components of the bandage.

Smith et al., U.S. Pat. No. 6,821,479, teaches a method of preserving abiological material in a dry protective matrix, the matrix comprisingfibers such as electrospun fibers. One component of the fibers may bedextran, but homogeneous dextran fibers are not described.

Cochrum et al., U.S. Pat. No. 7,101,862, teaches hemostatic compositionsand methods for controlling bleeding. The compositions comprise acellulose containing article (e.g. gauze) to which a polysaccharide iscovalently or ionically crosslinked. The crosslinked polysaccharide maybe dextran. However, the compositions are not electrospun and exogenousclotting agents are not included in the compositions.

Wnek et al., U.S. Patent Publication No. 2004/0018226, discloses fibersproduced by an electroprocessing technique such as electrospinning. Thefibers comprise enclosures within the fibers for containing substancesthat are not miscible with the fibers. Dextran is not taught as a fibercomponent.

Fisher et al., U.S. Patent Publication No. 2007/0160653, teaches ahemostatic textile comprising hemostatic factors (e.g. thrombin,fibrinogen) but the fibers are formed from electrospun glass plus asecondary fiber (e.g. silk, ceramic, bamboo, jute, rayon, etc.)

Carpenter et al., U.S. Patent Publication No. 2008/0020015, teacheswound dressing comprised of various biodegradable polymers and hydrogelshaving allogenic or autologous precursor cells (e.g. stem cells)dispersed within the polymers. The polymers may be prepared byelectrospinning, and one polymer component may be dextran. However, thepolymers cannot be immediately soluble upon contact with liquid, as theymust provide a scaffolding for delivery of the cells over time, eventhough the polymers eventually biodegrade in situ.

Li et al., U.S. Patent Publication No. 2008/0265469, describeselectrospun nanofibers that may include dextran. However, the nanofibersare not described as readily soluble in liquids.

Eskridge et al., U.S. Patent Publication No. 2009/0053288, teaches awoven hemostatic fabric comprised of about 65% fiberglass yarn and about35% bamboo yarn. The fiberglass component may be electrospun, andhemostatic factors such as thrombin may be associated with the fabric,e.g. by soaking the material in a solution of thrombin. This documentindicates that dextran may be added as a hygroscopic agent.

There is an ongoing need to provide improved methods and means toinitiate blood clotting in wounds to stop or at least slow blood loss.In particular, there is an ongoing need to improve the capability toreadily promote hemostasis in severe wounds in a facile manner,especially under circumstances where immediate treatment by medicalpersonnel is limited or unavailable.

Bowlin et al., U.S. Patent Publication No. 2011/0150973, discloses amethod of delivering one or more agents of interest to a location ofinterest. The method includes applying or delivering to a location ofinterest a hemostatic product. The hemostatic product includeselectrospun dextran fibers that dissolve upon contact with liquid. Thehemostatic product also includes one or more agents of interestassociated with said electrospun dextran fibers. Applying or deliveringresults in dissolution of the electrospun dextran fibers in liquid atthe location of interest to thereby release the one or more agents ofinterest into the liquid.

SUMMARY OF THE INVENTION

An embodiment of the invention is directed to a hemostatic product thatincludes a dextran support, at least one hemostatic agent and at leastone hemostatic agent stabilizer. The at least one hemostatic agent isselected from the group consisting of thrombin and fibrinogen. The atleast one hemostatic agent is associated with the dextran support. Theat least one hemostatic agent stabilizer is associated with the at leastone hemostatic agent.

Another embodiment of the invention is directed to a method of inducinghemostasis in a wound. A hemostatic product is fabricated and suchfabrication includes providing a dextran support. At least onehemostatic agent stabilizer is mixed with at least one hemostatic agent.The at least one hemostatic agent includes at least one of thrombin andfibrinogen. The at least one hemostatic agent is associated with thedextran support. The hemostatic product is applied to the wound in whicha bodily fluid is present. The at least one hemostatic agent is releasedfrom the hemostatic product and the at least one hemostatic agentinduces hemostasis in the wound.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of embodiments and are incorporated in and constitute apart of this specification. The drawings illustrate embodiments andtogether with the description serve to explain principles ofembodiments. Other embodiments and many of the intended advantages ofembodiments will be readily appreciated as they become better understoodby reference to the following detailed description. The elements of thedrawings are not necessarily to scale relative to each other. Likereference numerals designate corresponding similar parts.

FIG. 1. Schematic of the electrospinning apparatus. The key elements ofthe electrospinning system include a high voltage power supply, a sourcereservoir for the polymer and a grounded mandrel. This system utilizes acylindrical target mandrel; however the electrospinning process can beadapted to produce much more complex shapes. Single and/or multiplepolymers can be independently or simultaneously delivered to theelectric field from one or more source reservoirs. Electrospinningdistinct and unique polymers from separate sources in a temporalsequence can be used to produce a laminated structure.

FIGS. 2 A and B. A, schematic of air brush based dextran processing; B,dextran fibers produced by electroaerosol processing. The amount ofmaterial depicted is probably enough material for about two hemostaticproducts. Note the loft of the material. An electric field was used totarget the dextran to the mandrel.

FIG. 3. Scanning electron micrograph of electrospun dextran fibers. Thenominal average cross sectional diameter of the individual fibers was 1micron, providing a large surface area.

FIG. 4A-E. Schematic representations of exemplary hemostatic productsformed form electrospun dextran fibers. A, hemostatic product withnon-permeable support material as a backing; B, hemostatic product withnet-like support material; C, hemostatic product with non-permeablebacking and a net-like support material holding the electrospun fibersin place on the backing; D, hemostatic product for delivery oftherapeutics to a deep wound; E, alternative embodiment of a hemostaticproduct for delivery of therapeutics to a deep wound.

FIGS. 5A and B. Changes in cytokine levels in animals exposed to thesalmon fibrinogen/thrombin hemostatic product. (A) Levels of IL-1β,IL-6, TNF-α, IFN-γ, IL-4 and IL-10 are shown as the log ratio of thecytokine level determined in blood drawn at the initial surgery toimplant the vascular port compared to peak levels following exposure.Changes were seen in both pro-inflammatory responses (IL-1β, IL-6,TNF-α, IFN-γ) and humoral responses (IL-4 and IL-10). (B) Changes in thecytokines within an individual animal show that initial exposure (firstarrow) and the subsequent intravenous infusion of proteins (secondarrow) elicited a response that could be detected in samples taken atthe next blood draw.

FIG. 6A-F. Qualitative assessment of immunoglobulin production by swinein response to salmon proteins by Western blotting. (A) PAGE of salmon(Sal), human (Hu) and swine (Sw) fibrinogen preparations andcorresponding Western blots with serum from two animals (B and C). Serumfrom pre-exposure and final euthanasia blood draws are presented inthese panels. IgG isotypes present in the serum were visualized byspecific HRP anti-swine IgG second antibodies and are detected asbinding to the proteins in the gel samples. Arrows indicate thepositions of the IgG heavy and light chains components in the swineprotein lanes which are also recognized by the 2nd antibody. Molecularweights are show to the left (kDal×10⁻³). (D) PAGE of salmon (Sal),human (Hu) and swine (Sw) thrombin preparations and correspondingWestern blots with serum from the same animals shown in (C and D). Inthese animals, thrombin was not recognized in E, but there is a faintreaction in the salmon protein lane in F (arrow). The camera in thedetection system detected the heavy swine thrombin protein on themembrane as a white band in F.

FIG. 7A-D. Time course of antibody development in animals exposed tosalmon thrombin/fibrinogen hemostatic products through the dermal patchprotocol. ELISAs were performed using anti-IgG reagents. The followingantigens were used as the targets in the ELISAs: (A) salmon fibrinogen,(B) salmon thrombin, (C) human fibrinogen and (D) human thrombin. Theincreases in absorbance observed at the later samples panels A, B, Coccurred following intravenous infusion of salmon proteins. Each curverepresents data from a different animal.

FIG. 8A-D. Time course of antibody development in animals exposed tosalmon thrombin/fibrinogen hemostatic products through the abdominalpatch protocol. ELISAs were performed using anti-IgG reagents. Thefollowing antigens were used as the targets in the ELISAs: (A) salmonfibrinogen, (B) salmon thrombin, (C) human fibrinogen and (D) humanthrombin.

FIG. 9A-D. Progression of dermal healing following full-thickness wound.Images from samples taken at 7 days from control (A) and salmonhemostatic product-treated (B) injuries show a fibrinonecrotic coagulumfilling the wound defect (*) and an epithelial cell projection towardswound center in both cases as wound healing progresses following initialclotting. (H&E staining, bars=100 um). Samples taken at 28 days fromcontrol (C) and salmon hemostatic product-treated (D) injuries showcomplete re-epithelialization by a hyperplastic and hyperkeratoticepidermis. (H & E staining, bars=100 um).

FIG. 10. Schematic of the coagulation cascade.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An embodiment of the invention is directed to stabilizers that are usedin forming a hemostatic product. The stabilizers enable the hemostaticproduct to resist degradation when exposed to elevated temperaturesand/or radiation such as is used to sterilize the hemostatic product.

While the invention is described as being used in conjunction with ahemostatic product, the concepts of the invention may also be used inconjunction with other hemostatic products. An example of one suchhemostatic product is gauze. To forms in which the gauze may be providedis in a pad or in a roll.

The hemostatic product is held in place for a relatively short portionof time over a wound where it is desired to stop the flow of blood fromthe patient. In certain embodiments, the relatively short period of timeis less than about 5 minutes. In other embodiments, the relatively shortperiod of time is about 3 minutes.

The hemostatic product may be used in trauma situations where thecondition of the patient must be stabilized until it is possible totransport the patient to a treatment facility that access to medicaltreatment equipment that is more advanced to the medical treatmentequipment available where the patient was injured.

When the hemostatic product is applied to the injury site, the materialsused to fabricate the hemostatic product dissolve to thereby release thematerials to the injury site and provide the hemostatic effect. Thestabilizers may enhance the ability of the hemostatic product todissolve when the hemostatic products are applied to the injury site.

Prior to use of the hemostatic product, it may be desirable for thehemostatic product to be carried by a person on whom the hemostaticproduct could potentially be used and/or by a person who couldpotentially use the hemostatic product.

While the hemostatic product is being carried prior to use, it may bechallenging to protect the hemostatic product from not experiencingtemperature variations that are present in the regions where the personis located. In certain embodiments, preventing degradation of thehemostatic product at conventional room is exhibited. In otherembodiments, the hemostatic product resists degradation at temperaturesof more than 140° F. to less than 0° F.

While the components used in fabricating the hemostatic product do nottypically experience degradation when exposed to low temperatures, it isdesirable to minimize degradation when the hemostatic product is exposedto elevated temperatures such as up to about 150° F., which is typicallya maximum temperature that a person carrying the hemostatic productwould be exposed to.

In certain embodiments, the hemostatic product should resist degradationwhen exposed to the elevated temperature for more than about 3 hours. Inother embodiments, the hemostatic product should resist degradation whenexposed to the elevated temperature for up to about 24 hours.

A threshold for the hemostatic product to be viewed as not experiencingdegradation is that the hemostatic product does not exhibit noticeablevisible physical changes when viewing the hemostatic product withoutmagnification. The hemostatic product should also not experiencenoticeable physical changes when the hemostatic product is examined withmagnification such as with a magnifying glass or a microscope.

The preceding characteristics should be displayed by the hemostaticproduct regardless of whether the hemostatic product is retained in thepackaging materials while exposed to the elevated temperatureconditions.

The stabilizer also enhances the usable shelf life of the hemostaticproduct. In certain embodiments, the stabilizer provides the hemostaticproduct with a shelf life of at least about 2 years. In otherembodiments, the hemostatic product exhibits a shelf life of at least 3years. As used herein, the term usable shelf life means that thehemostatic product does not exhibit noticeable degradation when viewedwithout magnification or with magnification such as a magnifying glassor microscope.

To minimize the potential of degradation of the hemostatic product, thehemostatic product should be protected from exposure to moisture becausewhen the components used in the hemostatic product are exposed tomoisture, the components degrade such as by dissolving.

The packaging in which the hemostatic product is placed provides amoisture barrier that prevents moisture from passing therethrough. Evenwhen the hemostatic product is exposed to elevated temperatures whenunpackaged, it is assumed that the moisture level in the air surroundingthe hemostatic product is relatively low.

In one embodiment, the site of action is a wound bed, and the activeagents that are delivered by the hemostatic product are factors oragents that participate in the coagulation cascade such as thrombin andfibrinogen. Application of the hemostatic product to a wound results indissolution of the dextran fibers in blood within the wound bed, whichin turn results in release or delivery of the active agents at or intothe site.

In one embodiment, the site of action is a wound bed, and the activeagents that are delivered by the hemostatic product are factors oragents that participate in the coagulation cascade such as thrombin andfibrinogen. Application of an electrospun dextran fiber hemostaticproduct to a wound results in dissolution of the dextran fibers in bloodwithin the wound bed, which in turn results in release or delivery ofthe active agents at or into the site.

Thrombin and fibrinogen that are associated with the hemostatic productare in forms that are biologically active when they come into contactwith blood. Hence, upon dissolution, the thrombin acts on thefibrinogen, converting it to fibrin, which then forms a clot within thewound to thereby staunch the flow of blood.

In some embodiments of the invention, only spun dextran fibers areutilized and thus after clot formation, there is no need to disturb theclot to remove hemostatic product components, since none remain at thesite. In other embodiments, as described below, the hemostatic productmay include other materials such as support or backing material, which,after initial rapid application of the hemostatic product, may later beremoved for further treatment of the wound by conventional methods.

Electrospinning is a non-mechanical processing strategy and can bescaled to accommodate the large volumes necessary to meet the needs ofcommercial processing. A schematic representation of one type of set-upfor electrospinning is provided in FIG. 1. In this process a polymersolution, or melt, is injected with current to create a chargeimbalance. The charged solution is then placed in proximity to agrounded target (in FIG. 1, a grounded mandrel).

At a critical voltage the charge imbalance begins to overcome thesurface tension of the polymer source, forming an electrically chargedjet. Within the electric field, the jet is directed towards the groundedtarget and the carrier solvent evaporates. Depending upon reactionconditions, and the polymers used in the process, electrospinning can beutilized to produce a fine aerosol of material or a continuous non-wovenmat of fibrillar material, as shown in FIG. 1.

For many polymers, the nature of the electrospinning processintrinsically provides a high degree of control over the diameter of theresulting fibers. Micron to nanoscale diameters can be selectivelyachieved simply by regulating the starting concentrations of thepolymers present in the electrospinning solutions. By controlling themotion of the ground target with respect to the source solution, fibrilsmay be deposited into a random matrix or into aligned arrays that areoriented along a defined axis.

A second schematic of an electrospinning apparatus is shown in FIG. 2A.The key elements of the electrospinning system include a high voltagepower supply, a source reservoir for the polymer and a grounded targetmandrel. The system that is depicted utilizes a cylindrical targetmandrel; however the electrospinning process can be adapted to producemuch more complex shapes.

Single and/or multiple polymers can be independently or simultaneouslydelivered to the electric field from one or more source reservoirs. Inaddition, electrospinning distinct and unique polymers from separatesources in a temporal sequence can be used to produce a laminatedstructure. FIG. 2B shows the result of electrospinning about 10 grams ofdextran dissolved in deionized water onto a round mandrel target, asdescribed in detail in the Example 1 below. FIG. 3 shows a scanningelectron micrograph of electrospun dextran fibers in which the averagecross sectional diameter of the individual fibers is about 1 micron.

Those of skill in the art will recognize that electrospinning is not theonly way to make dextran fibers. Such fibers may be produced by othermethods of aerosolization. However, the electric field helps in theefficient collection of the fibers, and electrospinning may yield moreuniform fibers. Other technologies which might also be employed forspinning dextran fibers, including those described in Luo et al., U.S.Pat. No. 7,067,444; Bogue et al., U.S. Pat. No. 6,116,880; and Fuisz etal., U.S. Pat. No. 5,447,423, the complete contents of each of which arehereby incorporated by reference.

In particular, so-called “cotton-candy machines” (with or withoutapplied electrostatic force) may be suitable for use in fabricating thedextran fibers of the invention. More detailed descriptions of methodsof preparing the dextran fibers of the invention are provided in Example2 below.

Other methods include compressing a dextran solution between two platesor other flat surfaces and drawing the plates or surfaces away from eachother, usually repeatedly. Dextran fibers form between the two surfaces.

In some embodiments, substances other than dextran are used to formfibers for use in the hemostatic products of the invention, especially(but not exclusively) when a cotton-candy machine is employed. Examplesof such substances include but are not limited to sugars such asdextrose, sucrose, etc.

The commercially available dextran that is used to produce theelectrospun fibers of the invention is synthesized from sucrose byenzymes on the cell surface of certain lactic acid bacteria, thebest-known being Leuconostoc mesenteroides and Streptococcus mutans.Dextran is a complex, branched glucan (a polysaccharide made of manyd-glucose molecules) composed of chains of varying lengths (e.g. from 10to 200 kilodaltons). The straight chain consists of α-1,6 glycosidiclinkages between glucose molecules, while branches begin from α-1,4linkages (and in some cases, α-1,2 and α-1,3 linkages as well).

Dextrans are commercially available in a wide range of molecular weightse.g. from about 10 kilodaltons (kDa) to about 200 kDa. Commercialpreparations are mixtures of dextrans of varying molecular weights,usually in narrower weight ranges and may be provided, for example, as“low” or “high” molecular weight dextrans. For example, “Dextran 40” hasan average molecular weight of 40 kDa, “Dextrans 75” has an averagemolecular weight of 75 kDa, etc.

In the practice of the invention, the dextrans used for electrospinningare typically in a molecular weight range of from about 10 to about 200kDa, or from about 25 to about 200 kDa, or from about 50 to about 200kDa, or from about 75 to 200 kDa, and usually from about 60 to 90 kDa,or from about 100 to about 200 kDa.

Further, as would be understood by those of skill in the art, the mediansize of the dextran molecules in a dextran preparation also has aneffect in that if the median weight is high in a particular lot, lessdextran may be used to form the desired amount of fibers.

In general, the conditions for electrospinning dextran are as follows:an ambient temperature of from about 60 to about 75° F., a relativehumidity of from about 30% to about 40%, and typically at least about20%. The resulting fibers are typically in the nanometer or millimeterrange of cross-sectional diameter, usually from about 0.75 microns toabout 1.25 microns.

The electrospun fibers are “dry” and should be protected from exposureto moisture to prevent premature dissolution. However, some water isassociated with the fibers and fiber compositions can contain from about7 to about 8% water, but must be less than about 5% when the fibers aresterilized by x-ray irradiation.

The hemostatic products of the invention are usually formed ofsubstantially homogeneous spun dextran. The amount of dextran perhemostatic product can vary widely, depending on the size of hemostaticproduct that is being manufactured, with typical hemostatic productformulations using from about 5-10 grams of dextran (usually100,000-200,000 Mr) per hemostatic product.

However, the range can be extended widely, e.g. from as low as about 0.5grams or less (for small hemostatic products) to as high as 100 or moregrams per hemostatic product, for large hemostatic products. In someembodiments of the invention, it may be helpful to use lesser amounts ofdextran (e.g. about 0.1 to about 0.5 grams of dextran per hemostaticproduct) to concentrate the active agents that are delivered by thehemostatic product into a smaller volume.

Of more consequence is the concentration of dextran in the solution fromwhich the fibers are spun. Generally, a solution of dextran forelectrospinning will be of a concentration in the range of from about0.1 to about 10 grams per ml of solvent, or from about 0.5 to about 5grams per ml, and usually such a solution is at a concentration of about1 gram per ml, ±about 0.15 mg. A preferred range would be from about 0.9to about 1.1 grams of dextran per ml of solution that is to be spun.

Those of skill in the art will recognize that, due to the variability ofmolecular weight ranges in dextran preparations, and due to inherentvariability from batch to batch of commercially available preparationspurporting to be of a particular molecular weight range, it is typicallynecessary to test each batch of dextran with respect to electrospinningproperties. Such tests are well within the purview of one of skill inthe art, and usually involve trials of electrospinning a range ofconcentrations of dextran dissolved in a suitable solvent, to ascertainwhich concentration(s) result(s) in the most desirable fibercharacteristics, e.g. stability (e.g. to heat, humidity, etc.),uniformity, cross-sectional diameter, etc.

Those of skill in the art will recognize that critical indicators ofsuccess are very obvious when trying a new batch of dextran. Too littledextran in the spinning solution results in “spitting” from the needle,while too much dextran results in the production of dried droplets, orfailure to spin at all.

Likewise, when the humidity is too low, similar results can occur, i.e.fibers fail to form and in some cases fail to target efficiently to theground. These characteristics can be assessed according to methods thatare well known to those of skill in the art, including but not limitedto visual observation, testing of fiber strength and flexibility,observation via electron microscopy, solubility testing, resistance toheat and/or irradiation, color and tendency to discoloration, etc. Aswould be understood by those of skill in the art, all such testing maybe carried out under varying conditions of heat, humidity, etc.Formulations may also be assessed using animal testing.

The area (length and width) of the hemostatic product of the inventioncan vary widely and can be adjusted by adjusting spinning parameters. Inaddition, the mats of dextran fibers can be cut to a desired size afterspinning Generally, the hemostatic product will be from about 0.5centimeters or less to about 30 centimeters or more in length and/orwidth, but larger or smaller sizes are also contemplated.

The height or thickness of the hemostatic product can likewise varyconsiderably depending on the intended use of the hemostatic product. Incertain embodiments, the hemostatic product has a thickness of betweenabout 1 millimeter and about 5 centimeters.

The thickness of the hemostatic product (which is related to the volume)may impact the rate of dissolution of the dextran upon contact withliquid. For example, a thin hemostatic product (e.g. about 2millimeters), will dissolve more rapidly than a hemostatic product thatis thicker, providing the loft of the fibers is comparable.

In most embodiments, dissolution of the dextran fibers is extremelyrapid, e.g. about 5 minutes or less after exposure to liquid, or about 4minutes or less, or about 3 minutes or less, or about 2 minutes or less,or about 1 minute or less, e.g. the hemostatic product typically takesonly a few seconds to dissolve (e.g. from about 1 to about 20 seconds todissolve.

This rapid dissolution may be referred to herein as “instantaneous” or“immediate” dissolution. Compression of an electrospun dextran mat maybe used to modulate the rate of dissolution, with greater levels ofcompression inversely impacting the rate, i.e. generally, the greaterthe degree of compression, the slower the rate of dissolution. The rapidrate of dissolution is advantageous, particularly when deliveringbiologically active agents (e.g. hemostatic agents) to a site of actionsuch as a wound. Rapid dissolution of the carrier dextran fibersprovides extremely rapid delivery of the hemostatic agents to the woundupon deployment of the hemostatic product.

Those of skill in the art will recognize that a plethora of liquidsolvents exist in which it is possible to dissolve dextran. However,superior results for electrospinning dextran are generally achieved whenthe solvent is water, especially deionized or distilled or deionized,distilled (ddH₂O) or other forms of relatively pure water. In addition,there is far less environmental impact associated with the use of water.

It has been found that, generally, high concentrations of salt in thesolvent should be avoided. Whereas salt is often used to facilitate thespinning of some electrospun polymers, this is not the case for dextran.The concentration of salts in the spinning solution should be kept at aminimum to successfully form dextran fibers.

The one or more active agents that are associated with the dextranfibers of the hemostatic product may be any active agent that it isdesirable or advantageous to deliver to the site where the electrospundextran fiber device is to be used or applied. In one embodiment of theinvention, the electrospun dextran fiber device is a hemostatic productand is used to deliver beneficial agents, for example, to a wound.

Such wounds include wounds and breaches of body or tissue integrity thatoccur as a result of trauma (e.g. accidental trauma, trauma resultingfrom conflicts such as gunshot wounds, knives, etc.), as well as woundswhich are purposefully incurred, such as surgical incisions, bodypiercings, etc.

Usually the agents are bioactive agents that have a beneficial ortherapeutic effect at the wound site. In one embodiment, the site is ableeding wound at which it is desired to form a blood clot to stop orslow the bleeding. In this embodiment, the therapeutic substances ofinterest may include, for example, thrombin and fibrinogen, althoughother agents active in promoting hemostasis, including but not limitedto capscian, may also be included.

In addition, electrospun or non-electrospun collagen, agents that absorbwater, various dry salts that would tend to absorb fluids when placed incontact with e.g. blood; engineered thrombin or thrombin mimics;engineered fibrinogen; agents that cause vasospasm (e.g. ADP,5-hydroxytryptamine, 5-HT and thromboxane, (TXA-2) to help contract andseal a bleeding vessel, etc. may also be included.

In addition, other components of the clotting cascade may be added tothe hemostatic product, for example: tissue factors that are normallyonly expressed on the surface of damaged cells and which start thenormal clotting cascade; serotonin which enhances platelet clumping andpromotes vessel constriction; and other agents that are used to replacemissing components of the clotting cascade in hemophilia, for example,factor 7 (which activates the so called external extrinsic coagulationcascade) and crude extracts of platelets. These agents essentially workto “jump start” clotting by initiating the cascade further down thereaction network, as illustrated in FIG. 10. In FIG. 10, the variousfactors (and their alternative nomenclature and/or characteristicsand/or activities) are as follows:

-   -   Factor VII (Proconvertin): serine protease, Vitamin K dependent        synthesis in the liver;    -   Factor VIII: Glycoprotein binds vWF, produced by endothelium and        liver;    -   Factor IX (Christmas-Eve Factor): serine protease;    -   Factor X (Stuart-Prowler Factor, Clotting Factor X): serine        endopeptidase, converts prothrombin to thrombin; and    -   Factor XI (Plasma thromboplastin antecedent): serine protease,        plasma protein;    -   Factor XII (Hageman factor): serine protease, plasma protein        binds collagen;    -   Factor XIII (Fibrin stabilizing Enzyme): stabilizes fibrin        polymer. plasma protein, also present in platelets and monocyte        linage.

In FIG. 10, italic pathways denote inhibition and the central role ofthrombin in the activation of coagulation and inactivation ofcoagulation processes is shown, where:

-   -   VI=Cofactor for Xa in the conversion of prothrombin to thrombin;    -   APC=Activated Protein C, an extracellular signal molecule,        inhibits FVI (equivalent to FVa, a cofactor of XA in the        conversion of prothrombin to thrombin) and FVIIIa through a        proteolytic event; and    -   TAFI=Thrombin Activatable Fibrinolysis Inhibitor, an inhibitor        of clot lysis.

In addition, active agents that function to promote late stages of woundhealing may also be included to, for example, facilitate cell migrationand remodeling. The incorporation of collagen is an example of such anactive agent.

One or more of any of these active agents may be used in the practice ofthe present invention. The therapeutic agents must be amenable to dryingand are associated with the other components of the hemostatic productin the dry state, since liquid may negatively affect at least one of thecomponents used in the hemostatic product. For example, the activeagents may be desiccated or lyophilized, or water may be removed by someother means.

Generally, the amount of water that is present in the substances whenthey are associated with the electrospun dextran fibers is less thanabout 5%, and preferably less that about 2%. These substances retainfull or partial activity when rehydrated, e.g. in blood. Generallytherapeutic substances associated with the hemostatic products of theinvention retain, upon contact with liquid, at least about 25%, or about50%, or even about 75 to 100% of their activity before drying ordesiccation, as compared to standard preparations of the substance usingstandard assays that are known to those of skill in the art.

In some embodiments, thrombin or fibrinogen, or both, are associatedwith the hemostatic product. In some embodiments, the thrombin andfibrinogen are salmon thrombin and fibrinogen. Advantages of usingsalmon as a source of these materials include but are not limited to thelack of concern about transmission of etiologic agents (e.g. viruses)that may occur when human and other mammalian sources of thrombin orfibrinogen (e.g. bovine) are used.

Salmon thrombin and fibrinogen are highly efficacious and have nodeleterious side effects, when used in the pig model, which is arecognized animal model that is considered to be indicative of resultsin humans.

The quantity of fibrinogen added to the hemostatic product is generallyin the range of from about 10 milligrams to about 3 grams. In certainembodiments, the amount of fibrinogen in each of the hemostatic productsis between about 20 milligrams to about 1 gram.

The quantity of thrombin added to each of the hemostatic products isgenerally between about 10 and 10,000 NIH Units. In certain embodiments,the amount of thrombin in each of the hemostatic products is betweenabout 20 and 6,000 NIH Units.

In some embodiments, the therapeutic agents may themselves beelectrospun. For example, the therapeutic agents are dissolved in andspun from a solution. In some embodiments, the therapeutic agents may beelectrospun into fibers. In other embodiments, the active agents may beelectrospun into other forms such as droplets, beads, etc.

In some applications, active agents such as thrombin may beelectrosprayed with sucrose to form sugar droplets, which tends tostabilize thrombin and can also “trap” other substances of interest fordelivery to the hemostatic product.

For thrombin and fibrinogen, in most embodiments, these (or other)active agents are in a finely dispersed dry, particulate or granularform e.g. as a fine powder or dust, as electrospinning may tend todecrease their activity. In other words, the active agents are notelectrospun either by themselves.

The provision of the substances in the form of a fine powder provides alarge surface area of contact for dissolution when the materials comeinto contact with fluid. Generally, such particles will have averagediameters of between about 1 and 10,000 microns, and, in certainembodiments, between about 10 and 1,000 microns.

Such dry solid particles may be formed by any of several means,including but not limited to grinding, pulverizing, crushing, etc.However, those of skill in the art will recognize that other forms ofthese active agents may also be included in the hemostatic product, e.g.flakes, films, sheets, strings, etc. Further, in some embodiments,thrombin and fibrinogen are in the form of electrospun droplets whenassociated with an excipient or carrier.

Association of substances of interest with the excipient or carrier maybe accomplished by any of many suitable techniques that are known tothose of skill in the art, and will depend in part on the precise formof the substance and the means at hand. For example, for powdered,particulate thrombin and fibrinogen, association may be carried out bysprinkling, shaking, blowing, etc. the agents onto a layer of theexcipient or carrier.

Depending on the density of the fiber mat, the substances of interestmay become relatively evenly dispersed throughout the woven mat offibers or may be largely confined to the topmost section of the fibermat. If no backing is present, the latter embodiment is preferable, toprevent the particulate substance of interest from falling through andout of the mat.

The density of the fibrous mat can be adjusted (e.g. increased), forexample, by adjusting its thickness and/or by compressing the mat underpressure so that the fibers are closer together. Other techniques forassociation also exist, e.g. the placement of dry but liquid solublesheets or strips of material onto or between layers of a carrier,electrospinning the added materials as a discrete layer or in discretelayers, etc., and any such technique may be employed.

The techniques for assembling the hemostatic products of the inventionmay be carried out manually or may be mechanized, or a combination ofmanual manipulation and mechanization may be used. For thrombin inparticular, 5,000 NIH Units of thrombin is a relatively small volume ofpowder. Therefore, inert carriers or bulking agents such as dextrose maybe added to insure more complete dispersal of active agents in thehemostatic product.

The association of substances of interest with the excipient may becarried out according to many different arrangements. For example, afirst layer of excipient may be formed, and one or more of thesubstances may be associated with the first layer. Then another secondlayer of excipient may be formed on top of the substance(s) of interest,and the same or other substances of interest may be associated with thesecond layer, and so on.

A final or outermost layer of excipient may be added to prevent thedislodgement of substances of interest from the layer(s) below. Thenumber of layers of excipient that are used in the hemostatic product ofthe invention may vary widely, from as few as 1-2 to as many as severaldozen, or even several hundred, depending on the desired characteristicsof the hemostatic product.

Typically, a hemostatic product will contain 1-2 layers. In otherembodiments the hemostatic product may include between 2-20 layers. Thevery slight amount of moisture that is present in a prepared hemostaticproduct may help to trap and retain the thrombin and fibrinogen on thesurface of the hemostatic product.

In some embodiments of the invention, the hemostatic products alsoinclude one or more support structures or support materials incorporatedtherein. For example, a backing may be incorporated into the hemostaticproduct.

The support material may be formed from various electrospun materialssuch as polyglycolic acid (PGA), polylactic acid (PLA), and theircopolymers (PLGAs); charged nylon, etc. In one embodiment, the supportmaterial is compressed electrospun dextran fibers. By “compressedelectrospun dextran fibers” we mean that electrospun dextran fibers arecompressed together under pressure.

Compression of electrospun dextran fibers is carried out, for example,under pressure between two plates (e.g. a vice), and can compress a matof fibers with a height (thickness) of about 3 inches to a sheet with aheight of about 0.5 inches or even less (e.g. about 0.1 to about 0.4inches). In some embodiments, the electrospun dextran fibers areelectrospun directly onto a previously electrospun support material,while in other embodiments, the support material and the electrospundextran fibers are associated after electrospinning of each, e.g. byjoining of one or more layers of each.

In other embodiments, the support material is not an electrospunmaterial but is some other (usually lightweight) material. Examples ofsuch materials include but are not limited to gauze; various plastics;hydrogels and other absorbent materials that can facilitate absorptionof blood and therefore clot formation; etc.

The support material may or may not be soluble in liquid, or may beslowly soluble in liquid, and may or may not be permeable to liquid.Slowly soluble materials include those from which absorbable ordissolving (biodegradable) stitches or sutures are formed, included PGA,polylactic and caprolactone polymers.

In certain embodiments, the support material may dissolve relativelyquickly such as less than about 1 hour. In other embodiments, thesupport material may dissolve within from about 10 days to 8 weeks. Ineither case, the support material provides the advantage of not havingto remove the hemostatic product and risk disrupting the clot.

However, in any case, the support material should not interfere with theimmediate dissolution of the excipients and delivery of the activeagents associated therewith into the liquid that dissolves theexcipients. Thus, the support material might be only on one side of theelectrospun dextran fiber device, so that when the device is, forexample, a hemostatic product, and is applied to a wound, the hemostaticproduct is oriented so that the excipients come into direct contact withthe blood in the wound bed and the support material does not, i.e. thesupport material is the “top” or outermost surface of the hemostaticproduct when placed on the wound.

This embodiment is illustrated, for example, in FIG. 4A, in whichelectrospun dextran fibers 10 are shown as deposited onto non-porous,liquid impermeable support material 20. When applied to a wound,excipient 10 would face downward into the wound, and non-porous supportmaterial 20 would face away from the wound.

This arrangement could provide an advantage in that pressure could beapplied to the wound through the support material, to facilitate thestoppage of bleeding. Alternatively, the support material may containpores, openings or spaces that allow liquid to access the excipients inthe hemostatic product even when the support material is present. Forexample, the support material may be a net or web of material that isinsoluble (or slowly soluble) but that permits liquid to freely accessthe excipients and associated substances of interest.

This embodiment is illustrated schematically in FIG. 4B, which showselectrospun dextran fibers 10 deposited on (or possibly under, or on andunder, or woven throughout) netting 40, which is shown partially inphantom where covered by electrospun dextran fibers 10. In yet otherembodiments, both a “backing” or “top” support material and a secondweb-like support material may be present in the devise.

This embodiment is illustrated schematically in FIG. 4C, which showselectrospun dextran fibers 10 deposited on non-porous support material50 and overlaid with net-like material 60, i.e. electrospun dextranfibers 10 are “sandwiched” between non-porous support material 50 andnet-like material 60.

One of skill in the art will be able to envision many other combinationsand shapes of excipient layers and support materials that would provideadvantages in particular scenarios. For example, excipients might bewrapped or wound around an elongated support such as a filament orstring, or wrapped around a particular form with the shape of a cavityin which the hemostatic product is likely to be placed, such as a bullethole, etc.

The crux of the problem at the site of a penetrating injury is that thewounded tissue is relatively inaccessible. For example, for a bulletwound (e.g. in the leg or thigh) bleeding does not occur as much at thesurface but deeper within the tissue, within a cavity formed by thebullet, where it cannot be easily treated by a hemostatic product thatis simply spread over the external site of the injury (e.g. the point ofentry of the bullet, knife, shrapnel, sword, bayonet, etc., or othercause of injury).

This aspect of the invention solves the problems associated withpenetrating injuries, which can cause extensive bleeding in the deeptissues, and takes advantage of the highly soluble nature of the dextranhemostatic product. A complicating factor in this type of injuryconcerns the ability to deliver hemostatic materials that are highlysoluble to such a site.

There may be bleeding and other fluids evident at the entry site of thewound and the application of a hemostatic product to this superficialsite may result in the complete dissolution of the hemostatic product atthe surface-without the delivery of the active materials to theunderlying source of the bleeding within the wound cavity. The inventioncircumvents this occurrence by providing delivery of active agents deepinto the wound. Prior art hemostatic products have failed to adequatelyaddress this problem.

The present invention solves this problem by providing a hemostaticproduct, the shape and application of that can be adapted to use withsuch wounds. For example, an elongated cylindrical “cigar-shaped”hemostatic product that contains thrombin and fibrinogen, and which maycontain support material, is provided.

The hemostatic product may be stored within a protective covering orpackaging or tube. This tube protects the hemostatic product from theambient environment. Both the hemostatic product and the tube arepreferably sterile. These components may be further enclosed in an outerwrapper of e.g. paper, polymer, blister pack, similar to that used fordisposable syringes, to prevent loss of sterility.

When used, the outer wrapping is torn open and the sterile tubecontaining the hemostatic product is accessed. In some embodiments, oneend of the tube is removed and placed over the outermost accessibleportion of the injury. The tube may also comprise a “plunger” or similarmeans which enables the user to expel the hemostatic product from thetube and into the wound, in effect “injecting” the hemostatic productinto the wound.

Means such as those that are used for the vaginal delivery of, forexample, tampons, (i.e. a “cylinder within a cylinder”) may be employed,or a syringe-like means of delivery may be used. The hemostatic productcan thereby be introduced deep into the tissue along the wound track andthe therapeutic agents in the hemostatic product are delivered to wherethey are most needed, i.e. to the interior of the wound.

The cylindrical configuration of the hemostatic product is also suitablefor use in situations where there is a large opening in tissue. In suchsituations, multiple cylindrical hemostatic products may be insertedinto the large opening to thereby fill a portion of such opening.

In other embodiments, a plunger per se is not included, but the tube isfashioned so that both ends can be opened, and the hemostatic can bepushed into the wound from one open end by exerting pressure on theopposite open end of the tube using any object that fits at leastpartially into the tube, sufficiently to push the hemostatic product outof the tube and into the wound. Examples of such objects include afinger and a stick.

Such an object may be included with the hemostatic product of theinvention. Those of skill in the art will recognize that, due to therelatively high malleability of some configurations, this embodiment ofthe hemostatic product may include support material around or within thehemostatic product (e.g. biologically compatible netting, rod, etc. thatwill disintegrate via biodegradation) to render the hemostatic productmore robust and less flexible as it is shunted down into the wound.

Further, the outermost end of the hemostatic product, that end on whichpressure is exerted (e.g. with a plunger) to expel the hemostaticproduct from the tube into the wound, may be reinforced with supportmaterial so that the plunger or other object used to push on thehemostatic product can deliver sufficient force to remove the hemostaticproduct from the tube.

An exemplary schematic depiction of this embodiment of the invention isprovided in FIG. 4D, where hemostatic product 100, comprised of spundextran fibers 110 and (optional) support material 120, and having afirst end 130 and second end 140 is illustrated as enclosed within tube200.

Hemostatic product 100 is enclosed within tube 200 but is not shown inphantom for the sake of clarity. Tube 200 has openings 210 and 220, bothof which may be capped prior to use (caps not shown) or may be leftopen, especially if the entire apparatus is packaged in sterilepackaging 400. Sterile packaging 400 is removed or breached to provideaccess the apparatus prior to use. To use the apparatus, openings 210and 220 of the tube must be open.

To deliver hemostatic product 10 to a penetrating wound, an object suchas plunger 300 in inserted into end 210 of the tube. Pressure is exertedon hemostatic product 100 as plunger 300 contacts device end 130, andhemostatic product 100 is consequently pushed out of tube 200 viaopening 220 (in the direction indicated by the arrows) and into thepenetrating wound (not shown). A second schematic representation of sucha hemostatic product is provided in FIG. 4E.

In this depiction, support material is not included and the dry, sterilehemostatic product material (e.g. dextran fibers) with associatedtherapeutic agents is located or positioned within a small, sealedcylinder with a cap at one end and a plunger at the other. Upondeployment, the cap is discarded, the open end of the cylinder is placedover the mouth of the wound and may be inserted into the wound, and theplunger is depressed, displacing or injecting the hemostatic productmaterial deeply into the wound.

Similar designs may be used to deliver the hemostatic product toorifices or channels such as the nasal passages, the ear canal, thevagina, the anus, into blood vessels, etc. The components that are usedin such an application will be formed into a shape that is on the orderof about 1 to about 6 inches in length, and from about ¼ inch to 1 inchin diameter, i.e. the dimensions of the hemostatic product will besuitable for insertion through the external opening and deep into anorifice or a wound cavity.

All such arrangements, shapes, and embodiments of carrier layers andsupport materials as described herein are intended to be encompassed bythe invention.

The hemostatic product may be sterilized prior to use, generally byusing electromagnetic radiation, for example, X-rays, gamma rays,ultraviolet light, etc. If thrombin is included in the hemostaticproduct, it may be desirable to reduce the moisture content of thehemostatic product (e.g. a bandage or gauze) to less than about 5%, topreserve thrombin activity during sterilization.

This moisture content reduction can be achieved by drying the fabricatedhemostatic product, e.g., under a vacuum, or by using a fabricationmethod that reduces moisture content from the beginning. Typically, thehemostatic products are sterilized using X-rays in a dose of betweenabout 5 and 25 kilograys (kGray). Any method that does not destroy thecarrier or the activity of substances associated with the fibers may beused to sterilize the hemostatic products of the invention.

When the hemostatic product is a bandage, the substances of interestthat are associated with the fibers of the hemostatic product mayinclude thrombin and fibrinogen, and the hemostatic product may be usedto staunch bleeding. However, the range of active ingredients may varywith the specific application of the hemostatic product.

For example, hemostatic products comprised of only thrombin might beused for small injuries or in combination with other interventions. Inaddition, other therapeutically beneficial substances may also beassociated with the hemostatic product, including but not limited to:antibiotics, medicaments that alleviate pain, growth factors, bonemorphogenic protein, vasoactive materials (e.g. substances that causevasospasms), steroids to reduce inflammation and combinations thereof.

In other embodiments, the devices of the invention do not contain agentsthat promote clotting. Those of skill in the art will recognize that thedevices of the invention are highly suitable for delivering manysubstances of interest to a desired liquid environment or location. Forexample, the devices may be designed for delivery of therapeutic orbeneficial substances to any moist environment of the body, where thereis sufficient liquid to dissolve the electrospun dextran fibers andrelease the active substance, and where dissolved dextran is notproblematic.

Examples include but are not limited to oral, nasal, tracheal, anal,lung, and vaginal delivery of substances such as anti-microbial agents,analgesic agents, nutritional agents, etc. Oral applications include thedelivery of substances useful for dental treatments, e.g. antibiotics,pain medications, whitening agents, etc. Examples of therapeutic orbeneficial substances that can be used in conjunction with the devicesinclude antibiotics, medicaments that alleviate pain, growth factors,bone morphogenic proteins, vasoactive materials (e.g. substances thatcause vasospasms), inflammation reduction steroids and combinationsthereof.

However, in some embodiments, no bodily fluid is present (or ifinsufficient body fluid is present) and the applied hemostatic productcan be “activated” by wetting, e.g. by spraying, or by otherwiseapplying a source of moisture (e.g. by exposing the hemostatic productto a moist material such as a sponge), or dropping hemostatic productsinto a liquid (e.g. a body of water), to cause release of the agents ofinterest associated with the dextran fibers.

Due to the small footprint and light-weight characteristics of thehemostatic products, they are ideal for situations where space andweight of supplies are at a premium. Examples of such situations includebut are not limited to: military operations where the weight and size ofthe components of a soldier's gear are an issue; in first aid kits; foremergency care during travel (e.g. during space flight, camping, etc.);etc.

The hemostatic products may be used in a variety of situations and for avariety of purposes in which space and weight are not considerations.For example, the hemostatic products of the invention provide aconvenient means to administer thrombin and fibrinogen to surgicalwounds in a conventional operating theater.

The hemostatic products of the invention may also be advantageouslyutilized whenever it is desired to package and eventually release one ormore dried substances, but where it is unfeasible or undesirable tohandle the dried substances directly, e.g. where the quantity isextremely small, or the substance is toxic.

In such cases, the electrospun dextran fiber hemostatic products of theinvention may serve as a “scaffolding” or carrier for containing,storing and/or transporting the substance(s) until use, i.e. untilcontacted with liquid that dissolves the electrospun dextran fibers,concomitantly releasing the substances into the liquid. Such substancesmay include, for example, enzymes or their precursors (e.g. pro-enzymesor zymogens) and their substrates, substances that activate a protein orenzyme (e.g. proteases, cofactors, etc.), and the like.

The invention also relates to the use of stabilizers that resist thepremature degradation of the components utilized in the hemostaticproduct. One such stabilizer is adapted for use in conjunction withthrombin.

It is believed that the thrombin stabilizer gets into the structure ofthe thrombin and thereby reduces the rate at which the thrombinbreakdowns. The at least one thrombin stabilizer is mixed with thethrombin before the thrombin is associated with the dextran.

In one embodiment, the thrombin stabilizer contains sucrose. In certainembodiments, the sucrose is used in the thrombin stabilizer at aconcentration of up to about 5 percent by weight of the thrombin. Inother embodiments, the sucrose concentration is about 1 percent byweight of the thrombin.

Prior to mixing the thrombin stabilizer with the thrombin, the thrombinstabilizer may be mixed with dextran. It is believed that the dextranenhances the ability of the sucrose to enter into the structure of thethrombin. In certain embodiments, the dextran is used in the thrombinstabilizer at a concentration of up to about 5 percent by weight of thethrombin. In other embodiments, the dextran concentration is about 1percent by weight of the thrombin.

Similarly, a stabilizer may be used in conjunction with the fibrinogen.Prior to applying the fibrinogen to the other components of thehemostatic product, the fibrinogen stabilizer may be mixed with thefibrinogen. It is believed that the fibrinogen stabilizer gets into thestructure of the fibrinogen and thereby reduces the rate at which thefibrinogen breaks down.

In one embodiment, the fibrinogen stabilizer contains sucrose. Incertain embodiments, the sucrose is used in the fibrinogen stabilizer ata concentration of up to about 5 percent by weight of the fibrinogen. Inother embodiments, the sucrose concentration is about 1 percent byweight of the fibrinogen.

Prior to mixing the fibrinogen stabilizer with the fibrinogen, thefibrinogen stabilizer may be mixed with a solubility enhancing agent. Itis believed that the solubility enhancing agent enhances the ability ofthe sucrose to enter into the structure of the fibrinogen.

In certain embodiments, the solubility enhancing agent is a detergent.In other embodiments, the solubility enhancing agent is a non-ionicsurfactant such as Pluronic, which is available from BASF.

In certain embodiments, the solubility enhancing agent is used in thefibrinogen stabilizer at a concentration of up to about 1 percent byweight of the fibrinogen. In other embodiments, the solubility enhancingagent concentration is about 0.002 percent by weight of the fibrinogen.

In another embodiment of the invention, the fibrinogen and thrombin areplaced on the surface of and/or integrated into the matrix of adissolving film. Using the fibrinogen and thrombin in such aconfiguration enables the hemostatic product to be positioned over theposition on the person's body where the blood is being emitted and, assuch, where hemostasis is desired.

The dissolvable film may be configured to dissolve relatively quicklywhen exposed to liquid such as blood. In certain embodiments, the filmdissolves in less than about 30 seconds. In other embodiments, the filmdissolves in less than about 5 seconds. An example of one suitabledissolving film is marketed by Hughes Medical Corp.

An example of another dissolving film is a dissolving paper that isfabricated from materials that do not pose a health hazard to thepatient after the dissolving paper has dissolved. In certainembodiments, the dissolving paper may be fabricated from a material thatenhances the ability of at least one of the fibrinogen and thrombin toachieve hemostasis. An example of one such dissolving paper is marketedby Daymark Technologies.

In another embodiment, the fibrinogen and thrombin are provided betweentwo layers of a dissolvable material. An example of one such suitabledissolving film is marketed by Hughes Medical Corp. and which isdiscussed above.

The fibrinogen and thrombin may be provided in a variety ofconfigurations using the concepts of the invention. In one suchconfiguration, at least one of the fibrinogen and the thrombin areprovided in a powder that is retained between the layers of thedissolvable material.

The dissolvable material should have sufficient structural integrity toretain the fibrinogen and thrombin therebetween while resistinginteraction with the fibrinogen and thrombin. The dissolvable materialshould also dissolve relatively quickly when exposed to liquids such asblood such that the fibrinogen and thrombin are released therefrom.

As used herein, “quickly dissolving” means that the dissolvable materialbreaks down to a sufficient extent such that a significant portion ofthe fibrinogen and thrombin are in contact with the blood in less thanabout 30 seconds. In other embodiments, the dissolvable material breaksdown in less than about 10 seconds.

The dissolvable material should also facilitate readily bonding suchthat two layers of the dissolvable material can be attached togetheraround the edges thereof to thereby form an enclosure that is adapted toretain the fibrinogen and thrombin therein.

An example of one suitable technique for attaching the dissolvablematerials to each other is applying a small amount of liquid to at leastone of the pieces of material that are intended to be bonded together.The water causes a slight breakdown of the dissolvable materials suchthat when two layers of the dissolvable material are placed adjacent toeach other, the layers of the dissolvable material bond together.

The dissolvable material may be fabricated from a variety of materials.The dissolvable material should not negatively impact the stability ofthe fibrinogen and thrombin. The material used to fabricate thedissolvable layer should also be selected to not have any adverse healtheffects on the person or animal on which the product is intended to beused.

In certain embodiments, the material used to fabricate the hemostaticproduct may alone or in conjunction with the fibrinogen or thrombinenhance the rate of hemostasis. Examples of components that may be usedfor the dissolvable material include cellulose derived materials.

In one configuration, a separate device is used to maintain thehemostatic product in a desired position with respect to the patient. Anexample of one such device is gauze that is wrapped around the portionof the body that is bleeding.

In another configuration, at least a portion of the hemostatic productis covered with an adhesive. The adhesive may be positioned around atleast a portion of an edge of the hemostatic product. In an alternativeconfiguration, the adhesive covers a substantial portion of an innersurface of the hemostatic product.

In such a configuration where the adhesive is likely to come intocontact with the portion of the patient's body that is bleeding, theadhesive should be selected to be biocompatible to minimize thepotential of the patient experiencing complications caused by contactbetween the adhesive and the tissue that is bleeding.

In another embodiment, the fibrinogen and thrombin may be placed insideof an enclosure and/or on the surface of an enclosure that does notdissolve when exposed to liquids such as blood. Such a configuration mayfacilitate forming a blood clot thereon such that the blood clot couldbe removed from the patient.

Such a configuration may be similar to a conventional tea bag in thatthe enclosure may be attached to a string that is used in conjunctionwith at least one of positioning the product proximate an area wherehemostasis is desired or removing the product and the associated bloodclot from the patient.

The string may be fabricated from a material that is sufficiently strongsuch that the string does not break either when positioning the productor when removing the product from the patient. The string should also befabricated from a material that is not likely to produce adversebiological interactions.

The enclosure may be configured to discharge the fibrinogen or thrombinover a selected period of time. The rate at which the fibrinogen andthrombin are discharged may be adjustable based upon the rate and/orvolume of blood that is being discharged from the patient.

In an alternative configuration, the enclosure may be configured tobreakdown over an extended period of time. As the enclosure breaks down,the fibrinogen and thrombin may be discharged from the product.

By controlling the rate at which the fibrinogen and thrombin aredischarged from the product and/or the rate at which the enclosuredegrades, the product minimizes the formation of a clot having arelatively large size but rather may facilitate the formation of aplurality of clots having a smaller size. Such smaller clots may be morereadily broken down within the body than if relatively large clots werecaused to be formed.

This configuration of the hemostasis product may be particularly suitedfor use in conjunction with bleeding in patients that while being withina bodily cavity such a bodily cavity is accessible from outside of thebody. Examples of surgical techniques with which the hemostasis productmay be used in conjunction include sinus and tonsil surgery.

In another embodiment, the fibrinogen and thrombin are compressed into atablet. In addition to the fibrinogen and thrombin, the tablet may alsoinclude at least one excipient. The excipient should facilitate not onlyholding together the fibrinogen and thrombin as well as promotingrelatively quickly dissolving of the tablet.

As used herein, the term “relatively quickly” means that the tabletsdissolve when placed in a liquid in less than about 30 seconds. In otherconfigurations, the tablets dissolve in less than about 10 seconds.Quickly dissolving the tablets enables the fibrinogen and the thrombinto be quickly released from the tablets such that these materials mayprovide rapid hemostasis.

Additionally, in certain embodiments, the excipients that are used informulating the tablets should not decrease the stability and/orsolubility of the fibrinogen and the thrombin. In certain embodiments,the excipients used in formulating the tablets should increase thestability of the fibrinogen and thrombin.

An example of one such excipient is sorbitol, which has been formed intosmall particles such as by using spray-drying. In one suchconfiguration, the particles have a generally spherical shape and have agenerally uniform size.

The spray-dried sorbitol particles not only provide advantageousflowability characteristics but also exhibit desirable compactabilitycharacteristics when forming the tablets using a direct compressiontechnique.

Additionally, the spray-dried sorbitol particles provide good solubilityfor release of the fibrinogen and thrombin from the tablets. An exampleof one such spray-dried sorbitol particle is marketed by SPI Pharmaunder the designation SORBITAB SD 250.

Another excipient that may be used in fabricating the tablets ismannitol, which has been formed into small particles such as be usingspray drying. The particles may be formed with a narrow particle sizedistribution, which reduces the potential of the components segregatingwhile the tablets are being formed.

An advantage of the mannitol is that this material is non-hydroscopicsuch that the mannitol does not add moisture to the other componentsused in the tablets or contribute to moisture pickup either during theprocess of forming the tablets or after the tablets have been formed.The mannitol thereby protects the water-sensitive fibrinogen andthrombin.

The spray-dried mannitol particles not only provide advantageousflowability characteristics but also exhibit desirable compactabilitycharacteristics when forming the tablets using a direct compressiontechnique.

The spray-dried mannitol particles also promote rapid disintegration ordissolvability of the tablets such that the fibrinogen and thrombin canbe quickly released from the tablets. An example of one such spray-driedmannitol particle is marketed by SPI Pharma under the designationMANNOGEM EZ.

Other materials that may be used as excipients when preparing thetablets include fructose and maltose. Similar to the other excipientsthat are discussed above, the preceding excipients may be formed intosmall particles before being used mixed with the other components thatare used in the tablets.

Another excipient that may be used in conjunction with fibrinogen andthrombin is a quick dissolving platform that is marketed under thedesignation PHARMABURST 500 by SPI Pharma. This material provides thetablets with the ability to be rapidly dissolved while also providingdesirable characteristics for compaction and friability.

Depending on the excipient that is used in the tablet, it may also bedesirable to use a lubricant when preparing the tablet. The lubricantmay enhance the physical properties of the tablets. Examples of suchphysical properties include brittleness, friability and hardness. Anexample of one such lubricant is sodium stearyl fumarate, which isavailable from SPI Pharma under the designation LUBRIPHARM.

The concentration of the lubricant that is used in fabricating thetablets may depend on a variety of factors such as the types ofexcipients that are used. In certain embodiments, the concentration ofthe lubricant is up to about 5 percent by weight. In other embodiments,the concentration of the lubricant is between about 2 and 3 percent byweight. In still other embodiments, the concentration of the lubricantis about 2.5 percent by weight.

Once the components are mixed together, the mixture is subjected tocompression, which thereby causes the components to form the tablets. Incertain embodiments, the compressive force is at least 5,000 psi. Inother embodiments, the compressive force is between about 10,000 psi andabout 12,000 psi.

When preparing the tablets using the preceding process, it may not benecessary to include dextran. Even though dextran may not be required,it is possible to use dextran along with the other components that areused to formulate the tablets.

In another embodiment of the invention, the fibrinogen and thrombin maybe incorporated into a fast dissolving tablet such as by usingtechnology marketed by Catalent Corporation under the designation Zydis.

The fast dissolving tablets dissolve in less than 30 seconds and, insome configurations, dissolve in less than about 5 seconds. Quicklydissolving the tablets is important because at the tablets dissolve, thefibrinogen and thrombin contained therein is released and can therebyproduce hemostasis.

The amount of the fibrinogen and thrombin used in the tablet may beselected based upon the volume of bleeding. In certain embodiments,there is up to about 1 gram of fibrinogen and thrombin in each of thetablets. In other embodiments, there is about 500 micrograms offibrinogen and thrombin in each of the tablets.

In another embodiment of the invention, the fibrinogen and thrombin areapplied to a surface of or incorporated into an applicator. Such anapplicator enables the fibrinogen and thrombin to be accuratelydelivered to an area where hemostasis is desired.

In one such configuration, the applicator has an elongated portion thatmay be grasped by a person who is using the hemostatic product. Theapplicator may have a configuration that is similar to a swab. Thisconfiguration of the hemostatic product is particularly suited forlocations that are difficult to directly reach. An example of one suchcondition that this hemostatic product may be used to treat isepistaxis.

At least one of the fibrinogen and thrombin may be electrospun eitheralone or with another component such as dextran. The fibers producedusing such a process may be wrapped around a distal end of theapplicator.

The applicator may be configured to release the fibrinogen and thrombinonce the hemostatic product encounters blood. Using such a process, thefibrinogen and thrombin would cause clots to form. The clots could beremoved from the patient. If the clots are sufficiently small, the clotsmay be allowed to remain in the patient such that the clots couldeventually be broken down.

In another configuration of this hemostatic product, at least one of thefibrinogen and thrombin may be configured to remain relatively close toor be confined to the applicator such that when the fibrinogen andthrombin cause at least one clot to form, such clots remain attached tothe applicator. This configuration facilitates removal of the clots fromthe patient and may be desirable where the clots are likely to besufficiently large to make it undesirable for the clots to remain in thebody.

To facilitate the fibrinogen and thrombin not being released from theapplicator, the fibrinogen and thrombin may be incorporated into amaterial that is attached to an end of the applicator. An example of onesuch material is foam. The foam may be either open cell foam or closedcell foam. The foam should have pores that are sufficiently large toreceive the fibrinogen and thrombin. The foam should not have a strongaffinity for either fibrinogen or thrombin so that when the fibrinogenand thrombin are exposed to water, these components are released fromthe foam.

In another embodiment of the invention, the fibrinogen and thrombin areincorporated into foam. An example of one such suitable foam is anabsorbable gelatin sponge such as is available under the designationVETSPON from Novartis.

Depending on the application at which it is desired to use thehemostatic sponge, it may be desirable to prewet the hemostatic spongeprior to the hemostatic sponge being applied to the region wherehemostasis is desired.

Another advantage of using the foam is that the foam may be configuredto be bendable so that the hemostatic foam can be bent into aconfiguration that conforms to the shape of the region in which thehemostasis is desired. Once the hemostatic foam is bent into the desiredconfiguration, it may remain in that configuration even without afastening device being used to hold the hemostatic foam in the desiredshape and/or position.

Similar to the foam that is described above, the foam may be either opencell foam or closed cell foam. The foam should not have a strongaffinity for either fibrinogen or thrombin so that when the fibrinogenand thrombin are exposed to water, these components are released fromthe foam.

The fibrinogen and thrombin may be incorporated into the components thatare used to fabricate the foam such that rather than the fibrinogen andthrombin being applied to a surface of the foam, the fibrinogen andthrombin are dispersed through the matrix of the foam.

Such a configuration facilitates ongoing release of the fibrinogen andthrombin from the foam and may be particularly beneficial when it isdesired to form a clot in a region of the body that is likely toexperience rebleeding.

The quick dissolving tablets are suited for use in a variety ofapplications. An example of one such application is oral bleeding. Ifthe product is intended for use in conjunction with oral bleeding, thetablet may be flavored.

In conjunction with various surgical techniques, it is necessary to forman incision in the patient. Once the surgery is complete, it isnecessary for sutures or staples to be used to close up the incision.While the sutures or staples are effective at holding together thetissue, these closure mechanisms are not always effective to stop bloodfrom flowing out through the incision.

The hemostatic product may be placed over at least a portion of thesuture line through which blood is passing. The fibrinogen and thrombincontact such blood and thereby provide hemostasis.

Vascular access devices are used in conjunction with a variety ofmedical treatments such as delivering chemotherapy drugs to a patient.In one configuration, the vascular access devices are surgicallyimplanted in a large vein that is near the patient's heart. The vascularaccess devices may be left in place for an extended period of time suchas more than a year.

One challenge of surgically implanting the vascular access devices isstopping the bleeding around the vascular access device. For example,the blood may leak out of the suture lines or around the conduit. Insome situations where it is not possible to stop the leak, it isnecessary to bring the patient back to the operating room in an effortto stop the leak.

The hemostatic product may be placed around the vascular access deviceto thereby cause hemostasis. Depending on the shape of the vascularaccess device, it is possible for the hemostatic product to have avariety of configurations.

In other situations, the conduit associated with the vascular accessdevice may be porous. The hemostatic product may be used to providehemostasis and thereby prevent blood from passing through the porousconduit.

One or more of the preceding configurations of the hemostatic productmay be suited for use to stop bleeding from a patient such as when acatheter is removed from a femoral artery in the patient. In such aconfiguration, the hemostatic product may include an adhesive, whichholds the hemostatic product in place while hemostasis is occurring.

Aortic root surgery is used to treat a dilation or enlargement of theaorta. Because of the nature of the aorta, hemostasis plays an importantrole in the success of the procedure. One of the configurations of thehemostatic product that are discussed above may be used to provide thehemostasis without causing constriction of the aorta, which frequentlyresults from the prior art agents that are used to provide hemostasis inconjunction with this type of surgery.

For wounds that are deeper into the patient, it may be desirable to usethe hemostatic product in the form of a pledget that is placed at leastpartially into the wound and which thereby causes hemostasis within thewound.

In yet another configuration, the hemostatic product may be provided inan elongated configuration such as a tampon-type shape. The hemostaticproduct could also be formed in the shape of a worm or rope. Suchconfigurations could be used for hemostasis after vaginal surgeries.These configurations could also be used in conjunction with an elongatedhole in the patient such as may be caused by a gunshot wound.

The facilitate administering the elongated hemostatic product, it may bedesirable for the hemostatic product to be stored in an applicator suchas a plunger. The plunger could be used to insert the elongatedhemostatic product into the wound in the patient.

In another configuration of the hemostatic product, each of thecomponents of the hemostatic product are package separately such as in asyringe. Such a configuration enables the components to be dispensed atdifferent rates so that the hemostasis may be customized to theparticular patient or for a particular type of wound. The applicator mayallow each of the components to be delivered alternatively orsimultaneously.

The hemostatic product may consist substantially of the fibrinogen,thrombin and a carrier that are adapted to be bioabsorbed.Alternatively, the fibrinogen and thrombin may be placed on an outersurface of the hemostatic product such that the hemostatic product wouldbe removed from within the patient after hemostasis is completed.

In yet another configuration, the fibrinogen and thrombin are deliveredvia an aerosol. The fibrinogen and thrombin may be provide in the formof microspheres that are capable of being dispensed using an aerosolcontainer.

It is also possible to use the hemostatic product in conjunction withrobotic surgical procedures. While robotics provide the ability forsurgical procedures to be performed by a surgeon who is at a locationthat is remote to where the patient is located, the robotics havecertain limitations. The hemostatic product may be used in conjunctionwith the robotic surgical procedures to provide hemostasis and therebyovercome such limitations.

In addition to being used to produce hemostasis in humans, the conceptsof the invention may be adapted for use in conjunction with otheranimals. Examples of such animals on which the invention can be usedinclude dogs and cats.

In another embodiment of the invention, an effective amount of water ismixed with dextran to form an aqueous dextran solution. Thereafter, theaqueous dextran solution is electrospun to form a dextran sheet.

The dextran sheet may be stored until it is desired to fabricate thehemostatic product. In one such configuration, the dextran sheet isrolled. Rolling of the dextran sheet not only reduces the area taken upby the dextran sheet while the dextran sheet is being stored but alsoreduces the potential that the dextran sheet will be damaged prior tofabricating the hemostatic product.

When the dextran sheet is being rolled, care should be exercised to notroll the dextran sheet too tightly because such a process would increasethe density of the dextran sheet. Alternatively, tightly rolling thedextran sheet may be desired to increase the density of the dextransheet prior to fabricating the hemostatic product.

The thrombin and fibrinogen are mixed together at the ratio discussed inthe other portions of this patent application just before it is desiredto fabricate the hemostatic product. The mixing should provide arelatively uniform dispersion of the thrombin and fibrinogen in themixture.

In certain embodiments, the thrombin is dispersed on the dextran sheetto provide a thrombin concentration of between about 2 and 200 NIH Unitsof thrombin per square centimeter of the dextran sheet.

In certain embodiments, the fibrinogen is dispersed on the dextran sheetto provide a fibrinogen concentration of between about 20 and 60 gramsof fibrinogen per square centimeter of the dextran sheet.

The dextran sheet is unrolled and the thrombin and fibrinogen mixture isdispersed over the surface of the dextran sheet. In certain embodiments,the thrombin and fibrinogen mixture is dispersed in a substantiallyuniform manner over the surface of the dextran sheet. This evendispersion is desired because it enables each portion of the hemostaticproduct to have a substantially hemostatic activity.

This process is repeated and the sheets are stacked until the hemostaticproduct exhibits a desired amount of hemostatic activity. In certainembodiments, the hemostatic product includes between about 2 and 20dextran layers.

The thrombin and fibrinogen mixture is not placed on the uppermost layerof the dextran sheet. Using this configuration, the thrombin andfibrinogen are located at an interior location in the hemostaticproduct. Fabricating the hemostatic product in this manner enhances theability to retain thrombin and fibrinogen inside of the hemostaticproduct even though the thrombin and fibrinogen are sprinkled on thesurface of the dextran sheet.

While it is possible to put thrombin and fibrinogen on the outside ofthe hemostatic product, a portion of the thrombin and fibrinogen maybecome dissociated from the hemostatic product prior to use. In view ofthe cost of thrombin and fibrinogen, it is desirable for substantiallyall of the thrombin and fibrinogen to remain associated with thehemostatic product until it is desired to use the hemostatic product tomaximize the efficacy of the hemostatic product.

Even though the thrombin and fibrinogen are mixed together prior toplacing the thrombin and fibrinogen on the dextran sheet, the thrombinand fibrinogen are sufficiently dispersed on the dextran sheet so thatthe thrombin and fibrinogen do not react prior to placing the hemostaticproduct at the location where hemostasis is desired.

The hemostatic product is then cut into pieces. In certain embodiments,the pieces may be formed in a generally square shape. The size of thepieces may be selected based upon the intended use of the hemostaticproduct. For example, when the hemostatic product is intended forsurgical applications, the pieces may have a smaller size than if thehemostatic product is intended for trauma applications.

A cutter may be used to cut the hemostatic product into the desiredsize. In addition to cutting the hemostatic product into pieces, thecutter may cause the layers of the dextran sheets that are adjacent tothe cutter to be compressed together. This compression causes thedextran layers to stay together.

In certain embodiments, the pieces of the hemostatic product are vacuumpackaged. In addition to maintaining the hemostatic product sterile, thevacuum packaging also compresses the layers in the hemostatic product,which enhances the ability of the layers to resist separation after thehemostatic product is removed from the package prior to use.

This process thereby enhances the ability to use the hemostatic productbecause the layers in the hemostatic product resist coming apart. Anadvantage of using this process is that no additional steps arenecessary to retain the layers together. Additionally, it does notrequire the use of additional components and/or additional processingsteps, which could affect the efficacy of the hemostatic product.

In another embodiment of the invention that is directed to fabricatingthe hemostatic product, dextran is mixed with water until asubstantially homogeneous mixture is prepared. The dextran may beprovided in a powder having a relatively fine particle granulation. Thedextran and water may be selected to have a relatively high purity suchas is typically used in medical applications. An example of one suchsuitable water is distilled water.

In one such configuration, there are between about 3 grams and about 9grams of dextran with about 6 milliliters of water. In otherembodiments, there are about 6 grams of dextran is mixed with about 6milliliters of water. A person of skill in the art will appreciate thata variety of techniques may be used to mix together the dextran andwater to produce the substantially homogeneous mixture from dextran andwater. A non-limiting example of a technique that may be used to mixtogether the dextran and water is electrospinning.

The duration of mixing that is needed to prepare the substantiallyhomogeneous mixture of dextran and water may depend on a variety offactors such as the type of equipment that is used to perform themixing. In certain configurations, this mixing is performed for greaterthan about 30 minutes. The mixing may be performed at a temperature ofbetween about 40° F. and about 70° F.

Next, thrombin is mixed with the dextran-water mixture. The thrombin isadded to the dextran-water mixture to provide the hemostatic producttherefrom with a concentration of thrombin that is between about 20 and6,000 NIH Units.

Thrombin may be provided in a powder having a relatively fine particlegranulation. The thrombin may be selected to have a relatively highpurity such as is typically used in medical applications.

Similar to the process used to prepare the dextran-water mixture,electrospinning may be used when mixing the thrombin with thedextran-water mixture. The mixing of the thrombin with the dextran-watermixture may be performed at a temperature of between about 40° F. andabout 70° F.

The duration of mixing that is needed to prepare the substantiallyhomogeneous mixture of thrombin, dextran and water may depend on avariety of factors such as the type of equipment that is used to performthe mixing. In certain configurations, the mixing is performed forbetween about 10 and 30 minutes. In other configurations, the mixing isperformed for about 1 hour.

Once the mixing is completed, the mixture is introduced into anelectrospinning machine. The electrospinning machine is configured toproduce a sheet having a width of up to about 1 meter. However, shorterwidths may also be used depending on the desired dimensions for thehemostatic product.

A release sheet is placed beneath the electrospinning machine onto whichthe fibers are placed. The release sheet provides support for the fibersduring the processing and cutting. The release sheet should be selectedto not interact with the components being used in the fabricating thehemostatic product. The release sheet may be configured to be separatedfrom the product after the cutting and/or other processing is completed.

Because of factors such as challenges associated with incorporating thefibrinogen with the other components utilized in forming the hemostaticproduct, the fibrinogen may be provided on a surface of one of thelayers in the hemostatic product as opposed to being incorporated intoone or more of the layers.

The product and method of the present invention are described in thefollowing examples. These examples are provided as an illustration ofthe invention and are not intended to limit the invention.

The product and method of the present invention are described in thefollowing examples. These examples are provided as an illustration ofthe invention and are not intended to limit the invention.

Example 1

The following tests were conducted to evaluate the dissolvability offibrinogen when used in conjunction with products produced according tothe techniques described herein. The dissolvability tests are alsoreferred to as hydration tests. While such tests do not replicate thein-vivo conditions, it is believed that these tests are indicative ofthe respective in-vivo dissolution rates.

Four samples were prepared using the processes set forth below. Thesamples included a control and three other tests that included thecomponents set forth below.

Sample 1, the control, was prepared by mixing fibrinogen with enoughwater to form a solution. The solution was lyophilized to produce apowder.

In addition to fibrinogen, Sample 2 includes sucrose at a concentrationof about 1 gram of sucrose for each gram of fibrinogen. The fibrinogenand sucrose were mixed with enough water to form a solution. Next, anon-ionic surfactant (Pluronic) was mixed into the solution at aconcentration of about 0.02 percent by volume. The solution waslyophilized to produce a powder.

In addition to fibrinogen, Sample 3 includes sucrose at a concentrationof about 2 grams of sucrose for each gram of fibrinogen. The fibrinogenand sucrose were mixed with enough water to form a solution. Next, anon-ionic surfactant (Pluronic) was mixed into the solution at aconcentration of about 0.02 percent by volume. The solution waslyophilized to produce a powder.

Sample 4 included fibrinogen mixed with enough water to form a solution.Sucrose was added to the solution at a concentration of about 20milligrams of sucrose per milliliter of solution. Dextran was added tothe solution at a concentration of about 20 milligrams of dextran permilliliter of solution. Next, a non-ionic surfactant (Pluronic) wasmixed into the solution at a concentration of about 0.02 percent byvolume. The solution was lyophilized to produce a powder.

For each of the dissolvability tests, approximately 125 milligrams ofeach of the fibrinogen powders produced in Example 1 was mixed withabout 5 milliliters of water in a small vial to form a mixture. Themixtures provided a fibrinogen concentration of between about 22 and 32milligrams per milliliter. The vials were placed on a roller plate andthe time for the fibrinogen to fully dissolve was recorded. Each of thetests was conducted three times. The averages of the three test resultsfor dissolvability are set forth in Table 1 below.

TABLE 1 Sample Time to dissolve (minutes) 1 (control) 93 2 60 3 33 4 22

Sample 1 exhibited the longest time for the fibrinogen to dissolve.

The addition of sucrose and Pluronic to Sample 2 provided a reduction ofthe time for the fibrinogen to dissolve of about 35 percent whencompared to the control, which was desirable.

The increased level of sucrose in Sample 3, when compared to Sample 2,provided a nearly 50 percent reduction in time for the fibrinogen todissolve when compared to Sample 2 and a reduction of time forfibrinogen to dissolve of more than 64 percent when compared to thecontrol.

The addition of sucrose, dextran and Pluronic to Sample 4 provide a timefor the fibrinogen to dissolve that was about 76 percent less than thecontrol, which was highly desirable.

Example 2

The following tests were conducted to evaluate the clot strength offibrinogen and thrombin when used in conjunction with products producedaccording to the techniques described herein. While such tests do notreplicate the in-vivo conditions, it is believed that these tests areindicative of the respective in-vivo clot strengths. Each of the testswas conducted three times. The averages of the three test results forclot strength are set forth in Table 2 below.

TABLE 2 Sample Clot strength (Pascals) 1 (control) 11,372 2 10,337 315,392 4 11,884

For Sample 1, the control, about 700 microliters of fibrinogen solution,which contains about 17.5 milligrams of the fibrinogen was mixed withabout 50 microliters of thrombin (containing about 15 NIH Units).

This mixture caused a clot to form that was placed into a Rheostressrheometer, which is available from Thermo Electron Corporation, tomeasure the clot strength. Sample 1 exhibited one of the lowest clotstrengths.

Sample 2 was prepared by mixing about 17.5 milligrams of sucrose intoabout 700 microliters of fibrinogen solution, which contained about 17.5milligrams of fibrinogen. A non-ionic surfactant (Pluronic) was added tothe mixture at a concentration of about 0.02 percent by volume. Next,about 50 microliters of thrombin (containing about 15 NIH Units) wasadded to the mixture, which caused a clot to form.

The sample was placed into a Rheostress rheometer to measure the clotstrength. The sucrose and Pluronic in Sample 2 caused a decrease in theclot strength of about 9 percent when compared to the control, which wasundesirable.

Sample 3 was prepared by mixing about 35 milligrams of sucrose intoabout 700 microliters of fibrinogen solution, which contained about 17.5milligrams of fibrinogen. A non-ionic surfactant (Pluronic) was added tothe mixture at a concentration of about 0.02 percent by volume. Next,about 50 microliters of thrombin (containing about 15 NIH Units) wasadded to the mixture, which caused a clot to form.

The sample was placed into a Rheostress rheometer to measure the clotstrength. The addition of the higher concentration of sucrose in Sample3 caused an increase in the clot strength of about more than 35 percentwhen compared to the control. This result is contrary to what would beexpected based upon the results from the addition of the lowerconcentration of sucrose in Sample 2, which caused a reduction in theclot strength compared to the control.

Sample 4 was prepared by mixing about 14 milligrams of sucrose and about14 milligrams of dextran into about 700 microliters of fibrinogensolution, which contained about 17.5 milligrams of fibrinogen. Anon-ionic surfactant (Pluronic) was added to the mixture at aconcentration of about 0.02 percent by volume. Next, about 50microliters of thrombin (containing about 15 NIH Units) was added to themixture, which caused a clot to form.

The sample was placed into a Rheostress rheometer to measure the clotstrength. Sample 4 also exhibited a clot strength that was about 4percent better than the control, which was desirable.

Even though Sample 4 exhibited a time for the fibrinogen to dissolvethat was slightly less than Sample 3, Sample 3 exhibited a clot strengththat was considerably higher than Sample 4. When balancing time todissolve and clot strength, which is important to prevent rebleeding, itis believed that Sample 3 would be superior to Sample 4.

Example 3

Dextran is mixed with an effective amount of water to form an aqueousdextran solution. The aqueous dextran solution is electrospun to form anelectrospun dextran sheet.

Thrombin and fibrinogen were mixed together and then dispersed on theelectrospun dextran sheet. The thrombin was dispensed at a rate ofbetween about 1.3 and 2.7 NIH Units per square centimeter of theelectrospun dextran sheet. The fibrinogen was dispensed at a rate ofbetween about 3.6 and 7.4 milligrams per square centimeter of theelectrospun dextran sheet

This process was repeated until there were 8 layers of the electrospundextran sheet in a stacked configuration. The thrombin and fibrinogenmixture was not dispersed on the surface of the uppermost layer. Theelectrospun dextran sheet has a thickness of between about 1 and 3millimeters.

A cutter was then used to cut the hemostatic product into pieces havinga width of about 4.8 centimeters and a length of about 4.8 centimeters.In addition to forming pieces of a desired size, the cutting causes theelectrospun dextran layers to be pushed together. This process causedthe electrospun dextran layers to resist separation. The pieces of thehemostatic product were vacuum packaged for storage until use. Inaddition to preventing contamination of the hemostatic product, thevacuum packaging caused the layers of the electrospun dextran to beurged together.

A commercially available absorbable fibrin sealant product marketedunder the designation TACHOSIL (Nycomed) was used to compare with theperformance of the hemostatic product described in this patentapplication.

The TACHOSIL product contains an equine collagen sponge that is coatedon one side thereof with the thrombin and fibrinogen. The TACHOSILproduct contains between 3.6 and 7.4 milligrams of human fibrinogen persquare centimeter of the product and between 1.3 and 2.7 NIH Units ofhuman thrombin per square centimeter of the product.

Utilizing the preceding information regarding the concentration ofthrombin and fibrinogen on the TACHOSIL product, the TACHOSIL productwas cut into square pieces to provide an amount of thrombin andfibrinogen on each piece of the TACHOSIL product that was approximatelyequal to the amount of thrombin and fibrinogen on the hemostatic productthat was prepared above.

The hemostatic efficacy of the hemostatic product of this patentapplication and the TACHOSIL product was evaluated using the swine liverinjury model, which has previously been used to evaluate hemostaticefficacy. The swine liver injury model enables multiple tests to beconducted on a single animal.

The tests were conducted using adult, domestic, breed-indifferent,female pigs having a weight of between about 50 and 70 kilograms. Eachanimal was subjected to an initial examination to verify the animal wasin good health. Prior to the procedure, the animals were quarantined forthree days during which the animals were provided food and water adlibitum.

Prior to initiating the procedure, anesthesia was administered to theanimal. During the procedure, anesthesia and fluid maintenance wasadministered to the animal. A heating pad was placed under the animalduring the procedure to assist in maintaining the animal's bodytemperature during the procedure.

A midline laparotomy was performed to provide access to the abdominalorgans where the hepatic biopsies were to be performed. A biopsy punchhaving a diameter of approximately 8 millimeters was used to create eachhepatic biopsy. The hepatic biopsies each have a width of about 8millimeters and a depth of about 4 millimeters. The cylindrical hepaticbiopsy was surgically removed with a scissor.

The presence of continuous free bleeding at the biopsy site was observedfor about 20-25 seconds. Thereafter, the hemostatic product was appliedto the biopsy site. The hemostatic product was held in place with manualpressure for about 20 seconds.

The manual pressure was then removed while leaving the hemostaticproduct on the biopsy site. The biopsy site was observed for about 2minutes for visible signs of active bleeding.

If bleeding persisted, an additional hemostatic product was placed overthe biopsy site. The hemostatic product was held in place with manualpressure for about 20 seconds. Thereafter, the manual pressure wasremoved while leaving the hemostatic product on the biopsy site. Thebiopsy site was observed for about 2 minutes for visible signs of activebleeding. This process was repeated for up to 4 times if the bleedinghad not previously stopped.

As a control, the biopsy punch was used to create a hepatic biopsyhaving a width of about 8 millimeters and a depth of about 4millimeters. The biopsy was observed for about 2 minutes. During thistime period, the biopsy did not stop bleeding.

Tests using the hemostatic product of this patent application, theTACHOSIL product and the control were performed in a random order tominimize the potential effect caused by the order in which thehemostatic product was used during the evaluation.

The order in which the evaluation of the hemostatic agents was performedand the results obtained from such evaluations are set forth in Table 1below.

TABLE 1 Number of compressions to Time to achieve Hemostasis agentachieve hemostasis hemostasis (minutes) Tachosil 2 4 This invention 1 2This invention 2 4 Control n/a n/a Tachosil 4 8 Control n/a n/a Thisinvention 1 2 Tachosil 2 4

The evaluation was repeated using 5 additional animals. The order inwhich the hemostatic products were used on the 5 additional animals wasalso randomized.

The results from the first evaluation set forth in Table 1 were combinedwith the results from the 5 additional animals. There were a total of 18samples for each the hemostatic product from this patent application andthe TACHOSIL product.

For these 18 samples, the mean time for the hemostatic product from thispatent application to cause hemostasis was 2.9 minutes with a standarddeviation of 1.7 minutes. For these 18 samples, the mean time for theTACHOSIL product to cause hemostasis was 4.7 minutes with a standarddeviation of 1.4 minutes. The hemostatic product from this patentapplication thereby provided a reduction in time to achieve hemostasisof nearly two minutes.

The animal blood was evaluated prior to the biopsy and after hemostasiswas achieved for potential changes in the hematology. There were nosignificant differences in the hematology results for the hemostaticproduct from this patent application and the TACHOSIL product.

The clots provided by the hemostatic product of this patent applicationwere compared to the clots provided by the TACHOSIL product usingpathology. Fibrin was identifiable in all of the biopsies treated withthe hemostatic product of this patent application. On the other hand,fibrin was not identifiable in 5 out of 18 biopsies treated with theTACHOSIL product.

The total number of biopsies needing additional compressions for thehemostatic product of this patent application was 5. The total number ofbiopsies needing additional compressions for the TACHOSIL product was17. The hemostatic product of this patent application thereby representsa reduction of biopsies needing additional compressions of over 70% ascompared to the TACHOSIL product.

The total number of additional compressions needed to provide hemostasiswith the hemostatic product of this patent application was 8. The totalnumber of additional compressions needed to provide hemostasis for theTACHOSIL product was 24. The hemostatic product of this patentapplication thereby represents a reduction of additional compressions ofover 60% as compared to the TACHOSIL product.

It is believed that the “basketweave” configuration of the hemostaticproduct of this patent application allowed entrapment of erythrocytesdispersed throughout the biopsy site. On the other hand, the TACHOSILproduct resulted in trapping of sheets of erythrocytes at the base ofthe biopsy site or between areas of amorphous implant material.

Because the thrombin and fibrinogen used in the TACHOSIL product areobtained from human plasma, there is a risk that the TACHOSIL productmay contain infectious agents. Another advantage of the hemostaticproduct of the current invention over the TACHOSIL is that thehemostatic product of the current invention appears to remain stable ata higher temperature than the TACHOSIL product.

While a direct comparison was not conducted, the electrospun dextranbase used in the hemostatic product produced according to this inventionrapidly dissolves upon contact with fluids. The dextran is absorbed intothe body in a relatively short time frame that ranges from minutes to afew days. The rapid depletion of the dextran carrier can providebeneficial results in conjunction with reducing the risk of inflammationand scaring.

In contrast, the collagen sponge used in the TACHOSIL product breaksdown over a much longer period of time such as up to about 4 months.This aspect of the TACHOSIL product is indicated by the manufacturer tobe a feature because it is indicated that the collagen sponge holdsfibrin clots to the wound surface to achieve hemostasis. The slowabsorption of collagen can be associated with increased risk ofinflammation and scaring.

The electrospun dextran used in the hemostatic product provides thishemostatic product with a higher level of flexibility compared to thecollagen sponge used in the TACHOSIL product. This enhanced flexibilityenables the hemostatic product described in this patent application tomore readily conform to the surface of the wound than the TACHOSILproduct.

In the preceding detailed description, reference is made to theaccompanying drawings, which form a part hereof, and in which is shownby way of illustration specific embodiments in which the invention maybe practiced. In this regard, directional terminology, such as “top,”“bottom,” “front,” “back,” “leading,” “trailing,” etc., is used withreference to the orientation of the Figure(s) being described. Becausecomponents of embodiments can be positioned in a number of differentorientations, the directional terminology is used for purposes ofillustration and is in no way limiting. It is to be understood thatother embodiments may be utilized and structural or logical changes maybe made without departing from the scope of the present invention. Thepreceding detailed description, therefore, is not to be taken in alimiting sense, and the scope of the present invention is defined by theappended claims.

It is contemplated that features disclosed in this application, as wellas those described in the above applications incorporated by reference,can be mixed and matched to suit particular circumstances. Various othermodifications and changes will be apparent to those of ordinary skill.

The invention claimed is:
 1. A hemostatic product comprising: a dextransupport; a hemostatic agent stabilizer mixture comprising sucrose anddextran; and a hemostatic mixture comprising the hemostatic agentstabilizer mixture and at least one hemostatic agent selected from thegroup consisting of thrombin and fibrinogen, wherein the hemostaticmixture is applied to the dextran support.
 2. The hemostatic product ofclaim 1, wherein the hemostatic agent stabilizer mixture is provided ata concentration of up to about 5 percent by weight of the hemostaticproduct.
 3. The hemostatic product of claim 1, and further comprising asolubility enhancing agent added to the hemostatic agent stabilizermixture.
 4. The hemostatic product of claim 3, wherein the solubilityenhancing agent comprises at least one detergent.
 5. The hemostaticproduct of claim 3, wherein the solubility enhancing agent is providedat a concentration of up to about 1 percent by weight of the hemostaticproduct.
 6. The hemostatic product of claim 1, wherein the hemostaticproduct does not exhibit degradation that is visible withoutmagnification when maintained at a temperature of less than about 120°F. for at least about 2 years.
 7. The hemostatic product of claim 1,wherein the dextran support comprises a plurality of electrospun dextranfibers.
 8. The hemostatic product of claim 1, wherein the hemostaticmixture is applied to the dextran support so that the fibrinogen isplaced on the dextran support at a concentration of between about 20 and60 milligrams per square centimeter of the dextran support, wherein thehemostatic mixture is applied to the dextran support so that thethrombin is placed on the dextran support at a concentration of betweenabout 2 and 200 NIH Units per square centimeter of the dextran support,or wherein the hemostatic mixture is applied to the dextran support sothat the thrombin and fibrinogen are placed on the dextran support in asubstantially uniform manner.
 9. The hemostatic product of claim 1,wherein the thrombin is salmon thrombin, wherein the fibrinogen issalmon fibrinogen and wherein the thrombin and the fibrinogen are inparticulate form.
 10. The hemostatic product of claim 1, wherein thehemostatic product further comprises a support material that isassociated with the dextran support, wherein the support material isselected from the group consisting of gauze, compressed electrospundextran, polyglycolytic acid polymers, polylactic acid polymers,caprolactone polymers, charged nylon and combinations thereof.